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Bordetella pertussis is the causal agent of whooping cough (pertussis), a highly contagious respiratory illness. This organism produces a group of virulence factors between which is possible to find to the adenylate cyclase toxin-hemolysin (CyaA) and pertactin (Prn). The first allows the bacteria inhibit phagocytosis and inflammatory response in the host, whereas the second is an adhesin that is frequently included as a component in acellular vaccines against B. pertussis. The objective of this work was to obtain Bordetella pertussis LPA311 mutants lacking adenylate cyclase toxin - hemolysin and largest producers of pertactin 2 with vaccinal purposes. The results revealed that in the seven mutants obtained the cyaA gene has been replaced by the prn2 gene. Additionally, one-way ANOVA showed that the levels of production of Prn in five of the mutants were significantly higher to the levels produced by the control (p < 0.05). These results showed that the strategy of replacement of the gene cyaA by the prn2 gene on B. pertussis LPA311, allows eliminating the CyaA expression and increasing the Prn production. [ABSTRACT FROM AUTHOR]

The adenylate cyclase - hemolysin toxin (ACT) is one of the main virulence factors in Bordetella pertussis. It was discovered in 1973 by Wolff and Cook as a contaminant in vaccine formulations against this microorganism. It belongs to the family of toxins that form pores at the plasma membrane of target cells and reaches the highest expression during the virulent phase of the bacteria. This toxin has 1706 aminoacid residues and is composed of two domains: the N-terminal domain, with calmodulin-sensitive adenylate cyclase activity and the C-terminal domain that promotes the formation of pores at the membrane of target cells. Furthermore, the integrins type αMβ2 molecules are natural receptors for the toxin, which binds to the N-glycosylated extracellular portion. ACT allows the bacteria to inhibit phagocytosis, activation of type 1 T-helper cells and cytotoxic T cells as well as gamma interferon production by macrophages. Despite of its toxic effects, the inactivated form of the toxin has been used in delivering immunogenic epitopes to antigen-presenting cells, in the identification of proteins secreted by the type III secretion system and in protein-protein interactions studies. [ABSTRACT FROM AUTHOR]

El artículo discurre sobre el aislamiento y la purificación de la toxina de bordetella pertussis y la tosferina. Los autores comentan sobre la producción de las vacunas antipertúsicas, la separación de las proteínas y electroforesis. Otros temas incluyen Western blotting y la detección de los anticuerpos.

Conventional vaccines are based on the application of diverse biochemical, microbiological and immunological methods over causal agents of diseases. Despite of success reached in vaccines for controlling and preventing a lot of infectious pathologies, in some other instances the results have not been rather fruitful, so that exploring new approaches and possibilities is imposed. The development and increase of the molecular biology means a revolution for designing new vaccines against a great variety of pathogenic microorganisms causing diseases that affect mankind and animals. From knowledge on whole genome sequences of these microorganisms in combination with new advanced technologies such as bioinformatics, DNA microarrays and proteomics it has been possible to identify new important antigens for the development of new vaccine candidates. The combination genomics - proteomics has rendered great advances in discovering "target" sites of vaccines against pathogenic bacteria. The ideal vaccine must be pathogen - specific, harboring conserved antigens in each strain, therefore having a universal cover, with the minimal number of antigens. Based on that knowledge, all antigens in silico may be predicted a priori, without growing the microorganism in vitro. This new approach has been successfully applied to identifying new antigens in Bordetella pertussis as well, which may be evaluated further for vaccine purposes, therefore representing a serious global effort, just to face whooping-cough or pertussis reemergence. [ABSTRACT FROM AUTHOR]

Bordetella pertussis is the causal agent of whooping cough (pertussis), a highly contagious respiratory illness. Although the initial vaccines against the disease consisted of killed cells, nowadays many countries use acellular variants less reactogenic and more efficacious. Pertactin (Prn) is one of the B. pertussis's antigens implicated in protective immunity of most of these acellular vaccines. Prn is a polymorfic autotransporter protein of around 60 kD and 13 different variants have been described so far; Prn1 and Prn2 are two of the most studied variants. For obtaining acellular vaccine components it is necessary to develop high-levels antigen expression systems. With this purpose, one of the strategies followed is increasing the rate of transcription for these protein's genes and their number of copies in the cell. In this work prn1 and prn2 genes were inserted into the broad-host-range plasmid pMMB67EH under the regulation of fhaB and tac promoters. As a result it was obtained B. pertussis strains capable of expressing between 11 and 23 times more Prn than analyzed wild ones. It was also obtained E. coli strains in which Prn was detected in association with the cellular surface. The detection and quantification of pertactin antigen was performed by ELISA. [ABSTRACT FROM AUTHOR]